CN113755338B - Polycyclic aromatic hydrocarbon degrading strain P.domesticum LJD-1, microbial inoculum and application thereof - Google Patents

Abstract

The invention discloses a polycyclic aromatic hydrocarbon degrading strain P.domesticum LJD-1 and a microbial inoculum and application thereof. The invention obtains a degradation strain LJD-1 which takes phenanthrene as a carbon source by domesticating and separating soil samples from a Hangzhou petroleum polluted site. LJD-1 can use phenanthrene as carbon source at an initial concentration of 50 mg. L^‑1After the inorganic salt culture solution is cultured for 7 days, the degradation rate can reach 79.1 percent, and after the inorganic salt culture solution is prepared into a microbial inoculum, the degradation rate is 61.0 percent. Therefore, the strain Pyrromema domesticum LJD-1 and the microbial inoculum thereof have strong repairing effect on phenanthrene. Therefore, the strain has good application potential in bioremediation of polycyclic aromatic hydrocarbons in petroleum-polluted soil.

Description

Polycyclic aromatic hydrocarbon degrading strain P.domesticum LJD-1, microbial inoculum and application thereof

The technical field is as follows:

the invention belongs to the field of organic pollutant degradation, and particularly relates to a polycyclic aromatic hydrocarbon degrading strain Pyrromema domesticum LJD-1, and a microbial inoculum and application thereof.

Background art:

with the rapid development of modern industry, the pollution caused by industrial wastewater is increasingly serious. Various organic contaminants, such as Polycyclic Aromatic Hydrocarbons (PAHs), are persistent. Polycyclic aromatic hydrocarbons are a class of hydrophobic organic compounds having two or more fused rings, which are ubiquitous byproducts of fossil fuels (coal, petroleum, etc.), waste incineration, and wood processing.

Bioremediation or microbial degradation of polycyclic aromatic hydrocarbons is the use of microorganisms to remove pollutants from wastewater, and is considered a cost-effective and eco-friendly remediation option and is therefore used as the primary treatment process in industrial sewage treatment plants. Phenanthrene is a tricyclic aromatic hydrocarbon, and has a very close relationship with carcinogenicity of PAHs. At present, few phenanthrene-degrading fungal strains have been reported, mainly including Trichoderma, Scedosporium, Fusarium, Penicillium, and Aspergillus, among others. Since most of the microorganisms in the environment are non-culturable, many microorganisms, especially those with specific functions, cannot be isolated by pure culture. Therefore, the screening of the bacterial strain capable of effectively degrading high-concentration phenanthrene has important application value and practical significance. The mass concentration of the test is 50 mg.L^-1The phenanthrene is used as a substrate for degrading the strain, so that data support is provided for biological treatment of the polycyclic aromatic hydrocarbon.

The invention content is as follows:

the first purpose of the invention is to provide a Pyrromema domesticum LJD-1 with polycyclic aromatic hydrocarbon degrading capability, which is preserved in Guangdong province microorganism culture Collection (GDMCC) at 7-12 months in 2021, with the address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No: 61797.

the reported strain in the research is Pyrromema soil and can be separated from soil. At present, the research on the degradation of the bacteria to pollutants is less, and related reports on the degradation of PAHs by the bacteria are not available at home and abroad. The research obtains 1 strain LJD-1 which takes high-concentration phenanthrene as a carbon source from a soil sample obtained from a certain petroleum-polluted site in Hangzhou through domestication and separation, and performs strain identification and growth characteristic research on the strain LJD-1. In addition, the strain is prepared into a microbial inoculum to explore the degradation characteristic of the LJD-1 microbial inoculum to phenanthrene, and a reference is provided for bioremediation of PAHs in petroleum-polluted soil.

The second purpose of the invention is to provide the application of Pyrromema domesticum LJD-1 in degrading phenanthrene.

Preferably, the degradation phenanthrene is phenanthrene in petroleum-polluted soil.

The Pyronema domesticum LJD-1 is preferably applied to degrade phenanthrene in a phenanthrene-polluted environment.

The third purpose of the invention is to provide a phenanthrene degradation microbial inoculum which contains Pyrromema domesticum LJD-1 as an active ingredient.

Preferably, the preparation method of the phenanthrene degradation microbial inoculum comprises the following steps:

a. heating corn and water according to a mass ratio of 1:5 to prepare paste, and mixing the paste according to a mass ratio of 150: adding wood dust, wheat bran and sodium lignosulfonate at a ratio of 100:10:1, kneading into a dough at a low temperature, putting the mixture of the dough-shaped culture matrixes into a pill kneading machine to prepare spherical culture matrixes, and sterilizing and drying for later use;

b. pyrromema domesticum LJD-1 is prepared into a bacterial liquid.

c. Adding the bacterial liquid into a sodium alginate solution with the mass fraction of 3% according to the mass ratio of 1:10, fully mixing the spherical culture medium with the solution, adding a sterile calcium chloride solution with the mass concentration of 4% after the mixture is completely mixed, and hardening to obtain the pellet-shaped encapsulated fungi;

d. and (3) putting the encapsulated fungus pellets into a sterile culture bag, culturing in an incubator at 28 ℃ for 3-7 days until white hyphae grow on the surface, thus obtaining the phenanthrene degradation microbial inoculum.

Preferably, the bacterial liquid in the step b is bacterial liquid with hypha content of 10 g/L.

The fourth purpose of the invention is to provide a method for degrading phenanthrene, which is to spray the Pyronema domesticum LJD-1 in an environment containing phenanthrene to degrade the phenanthrene.

Preferably, Pyronema domesticum LJD-1 is sprinkled in the environment polluted by phenanthrene to degrade the phenanthrene.

Preferably, Pyronema domesticum LJD-1 is sprinkled in the petroleum-polluted soil to degrade phenanthrene.

The invention obtains a degradation strain LJD-1 which takes phenanthrene as a carbon source by domesticating and separating soil samples from a Hangzhou petroleum polluted site, and the degradation strain LJD-1 can be obtained according to strain morphology, physiological characteristics,And (3) carrying out ITS gene sequencing analysis and phylogenetic analysis, and identifying the strain as Pyrromema domesticum LJD-1. The optimal environmental conditions for LJD-1 growth are: the temperature is 28 ℃, the pH is 6.0, and NaCl is not added; the ITS gene sequencing analysis result of the strain shows that the strain closest to LJD-1 is Pyrromema domesticum A10 (the similarity is 100%). LJD-1 can use phenanthrene as carbon source at an initial concentration of 50 mg. L^-1After the inorganic salt culture solution is cultured for 7 days, the degradation rate can reach 79.1 percent, and after the inorganic salt culture solution is prepared into a microbial inoculum, the degradation rate is 61.0 percent. Therefore, the strain Pyrromema domesticum LJD-1 and the microbial inoculum thereof have strong repairing effect on phenanthrene. Therefore, the strain has good application potential in bioremediation of polycyclic aromatic hydrocarbons in petroleum-polluted soil.

Pyrromema domesticticum LJD-1, deposited at 12 months 7 in 2021 at Guangdong province collection of microorganisms and cell cultures (GDMCC), address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No: 61797.

description of the drawings:

FIG. 1 shows the front and back sides of strain LJD-1 grown on PDA medium for 72 hours.

FIG. 2 shows the phylogenetic relationship of strain LJD-1 and related strains based on ITS gene sequences, which is constructed by the neighbor joining method, and the setting of the auto-unfolding value is repeated 1000 times, only the result that the auto-unfolding value is greater than 50% is shown in the figure, and the scale bar 0.01 represents the substitution rate of each nucleotide.

FIG. 3 shows the growth of strain LJD-1 under different conditions of temperature, pH and salinity tolerance, (a) pH; (b) (ii) temperature; (c) and (4) salinity resistance.

FIG. 4 shows the degradation efficiency of strains LJD-1 and LJD-1 (LGD-1-agent) in medium with inorganic salts of phenanthrene (initial concentration 50 mg. multidot.L)^-1)。

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1: isolation and characterization of Pyrromema domesticum LJD-1

1. Materials and methods

1.1 sample sources

Taking a soil sample from a certain petroleum-polluted site in Hangzhou, taking high-concentration phenanthrene as a carbon source for long-term domestication, and obtaining the efficient phenanthrene degrading bacteria through multiple screening, separation and purification.

1.2 culture Medium

1.2.1 inorganic salt Medium

The inorganic salt culture medium is used for enrichment culture of microorganisms in a sample, and phenanthrene degradation experiments under the conditions of pure bacteria and microbial agents. The medium formulation is shown in table 1. The preparation method comprises adding the above materials into solvent water, mixing, and sterilizing.

TABLE 1 inorganic salt media formulation

1.2.2 nutrient Medium

The nutrient medium is used for culturing conventional microorganisms such as separation, purification, preservation, activation and the like of fungi. The types and compositions of the liquid nutrient media used in this experiment are shown in Table 2. If a solid culture medium needs to be prepared in an experiment, only agar powder with the mass fraction of 1.5-2% needs to be added on the basis of the original culture medium formula. If the culture conditions of the strains are not specified, the pH of the culture medium is adjusted to 6. The preparation method comprises adding the above materials into solvent water, mixing, and sterilizing.

TABLE 2 Potatto-Dextrose broth (PDA) composition

1.3 acclimatization, screening and isolation of strains

Adding the collected contaminated soil into inorganic salt culture medium, adding streptomycin sulfate and penicillin (concentration is 100ug/ml) to inhibit fungus growth, and adding into the culture mediumAdding into the mixture at a concentration of 100 mg.L^-1The phenanthrene is used as a degradation substrate and is placed in an incubator at 28 ℃ and subjected to shake culture in the dark. 10% of the inoculum size was transferred to fresh 100 mg.L with the same culture system^-1And (3) in an inorganic salt culture medium with phenanthrene as a carbon source, repeating the enrichment process, wherein 7d is an acclimatization period, and repeating the process for three times.

And (3) coating and separating the fourth generation enrichment culture sample obtained by the method of dilution plate, and separating the sample by using nutrient medium. Culturing the coated sample at 28 deg.C for about 48 hr to form obvious single colony on the surface of the culture medium, selecting different single colonies according to the characteristics of colony, such as morphology, size, color, and hypha, streaking, purifying, and culturing. If single colonies of different characteristics are still observed on the streaked plates, they are streaked again until only single colonies of the same characteristics are observed on the same plate. 1 strain LJD-1 with high degradation performance to phenanthrene is obtained by screening in the experiment. Selecting purified single colony to culture in corresponding solid test tube nutrient medium, sealing with sterilized liquid paraffin, and storing at-4 deg.C for a long period.

A. Morphological characteristics

LJD-1 is a fungus separated from soil samples taken from an oil pollution site in Hangzhou, and can form a colony with a diameter of 8mm and a round shape with upward growth of white villous hyphae after being activated and growing on a PDA plate for 72 hours under the aerobic condition at 28 ℃ (FIG. 1). The strain is an obligate aerobic strain.

B. Characteristics of molecular biology

The molecular biological characteristic identification mainly comprises sequencing and the construction of a phylogenetic tree. Before sequencing and constructing phylogenetic tree, DNA of fungus needs to be extracted (the rapid extraction kit of the fungus genome DNA used in the experiment is from the company of biological engineering (Shanghai)). In order to study the taxonomy of fungi, it is usually necessary to amplify ITS gene, which is a piece of DNA in the eukaryotic coding rRNA component, and construct phylogenetic tree, which is usually used for detecting and identifying fungi due to ITS high degree of conservation, specificity and appropriate sequence length.

The Polymerase Chain Reaction (PCR) is mainly used for amplifying different gene fragments, the PCR requires different primers (ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'; ITS4: 5'-TCCTCCGCTTATTGATATGC-3'), and the PCR amplification reaction system: 10 XBuffer 2.5. mu.l, Mg²⁺1.5. mu.l (25mmol/l), 0.3. mu.l dNTP (25mmol/l), 0.5. mu.l forward primer (10mmol/l), 0.5. mu.l reverse primer (10mmol/l), Taq enzyme: 0.25. mu.l, 0.1. mu.l of DNA set template, 19.35. mu.l of deionized water. PCR amplification reaction conditions: pre-denaturation at 95 ℃ for 3min, annealing at 95 ℃ for 45s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 45s, for 30 cycles. Extending for 10min at 72 ℃, and storing at 4 ℃ after the reaction is finished. Amplifying the needed gene, adding 0.75-1% agarose and nucleic acid stain GelRed to prepare a gel block, adding PCR products and DNA markers (marker) containing fragments of various lengths into the gel block, placing the gel block into an electrophoresis apparatus, filling TBE (Tris boric acid) buffer solution into the electrophoresis apparatus, taking out the gel block after the electrophoresis apparatus works for 20min under a certain voltage, and placing the gel block under an ultraviolet lamp of 300nm for observation to confirm that the PCR product amplification reaction is successful. Then, the pcr product successfully amplified is sent to Huada Gene science and technology Limited for sequencing, and the sequencing primer is the same as the amplification primer.

The sequencing obtained fungal ITS gene sequence is uploaded to EzTaxon-e (http:// EzTaxon-e. ezbiocloud. net), the website compares the submitted sequence with the ITS gene sequence of a recognized typical strain to obtain the similarity information between the sequences, the corresponding typical strain can be selected as the model bacteria of the experimental isolate strain according to the analysis of the sequence comparison result, meanwhile, the ITS gene sequence of the model bacteria can be obtained, phylogenetic analysis is constructed to prove that the model bacteria and the experimental isolate strain have difference so as to identify the isolated strain, the phylogenetic tree is constructed by utilizing MEGA 5.05 program, the adjacency method, the minimum evolution method and the maximum reduction method are usually adopted to construct the evolutionary tree, wherein the most common adjacency method is the adjacency method, and the self-development value is usually set to repeat 1000 times of calculation.

The ITS gene alignment shows that the gene similarity of the strain LJD-1 (the nucleotide sequence of the ITS gene is shown as SEQ ID NO. 1) and Pyrromema domesticum strain A10 is 100%. From the above results, it was found that the fungus LJD-1 isolated in this experiment is Pyrromema domesticum.

And (3) making a phylogenetic tree by using the ITS gene sequence of the LJD-1 and the ITS gene sequence with higher similarity, thereby obtaining a homology result between the ITS gene of the LJD-1 and the ITS gene with higher similarity. A phylogenetic tree constructed by the orthotopic grafting method is shown in FIG. 2. At present, there are few reports about the application of the strain in the environmental field. Therefore, the obtained efficient phenanthrene degrading bacteria have important theoretical and practical significance for treatment and deep repair of PAHs contaminated soil.

From the above results, it was found that the fungus LJD-1 isolated in this experiment is Pyrromema domesticum. Designated as Pyrromema domesticticum LJD-1, deposited at the Guangdong province culture Collection (GDMCC) at 12/7/2021, address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No: 61797.

example 2: growth conditions of Pyrromema domesticum LJD-1

A. Measurement of growth temperature:

a liquid nutrient medium (PDA medium in example 1) required for growth of the strain was prepared, and the prepared liquid nutrient medium was sterilized in a sterilizer. Inoculating 10.1 ml of activated strain Pyrromema domesticum LJD-10.1 to 10ml of PDA culture medium (experimental group), taking the PDA culture medium without inoculated fungi as a control group, putting the culture medium into different temperatures for culturing for 7 days, repeating the control group and the experimental group corresponding to each temperature for three times, observing the growth condition of the fungi every day, pouring the culture medium into a weighing centrifuge tube after 7 days, centrifuging for 30min at 5000 revolutions, pouring out supernatant, putting into a 60-DEG oven, drying to constant weight, and weighing to calculate the dry weight of fungal hyphae. The test temperatures were as follows: 18 ℃, 23 ℃, 28 ℃, 33 ℃ and 38 ℃.

B. Measurement of growth pH:

preparing a liquid nutrient medium (PDA medium in example 1) required for strain growth, and adjusting the pH of the culture solution with the following buffer system, wherein the pH is 4.0-5.0, 0.1mol/l sodium citrate and 0.1mol/l citric acid; pH 6.08.0, 0.1mol/l NaOH and 0.1mol/l KH₂PO₄；pH 9.0，0.1mol/l NaHCO₃And 0.1mol/l Na₂CO₃. Inoculating 10ml of activated strain Pyrromema domesticum LJD-10.1 ml into 10ml of PDA culture media (experimental group) with different pH values, repeating three times for each pH value, using a culture medium without inoculated fungi as a control, putting the culture medium into an optimal temperature for new fungi growth for culturing for 7d, observing the growth condition of the fungi every day, pouring the culture medium into a weighed centrifuge tube after 7 days, centrifuging for 30min after 5000 turns, pouring off a supernatant, putting into a 60-DEG oven, drying to constant weight, and weighing to calculate the dry weight of the fungal hyphae. The pH tested was as follows: 4.0, 5.0, 6.0, 7.0, 8.0, 9.0.

C. Salt concentration tolerance: liquid nutrient medium (PDA medium of example 1) required for growth of the strain was prepared, and the salt concentration of the medium was adjusted. Inoculating 10.1 ml of activated strain Pyrromema domesticum LJD-into 10ml of PDA culture medium (experimental group) with different salt concentrations, repeating three times for each salt concentration, using a culture medium without inoculation as a control, placing the culture medium at the optimal temperature for new bacteria growth for culturing for 7d, pouring the culture medium into a weighed centrifuge tube after 7 days, centrifuging for 30min after 5000 turns, pouring out a supernatant, placing into a 60-DEG oven, drying to constant weight, weighing and calculating the dry weight of fungal hyphae to obtain the salt concentration range which can be tolerated by the new bacteria. The salt concentrations tested were as follows: the mass fraction is 0%, 2%, 4%, 6%, 8%, 10%.

As shown in FIG. 3, in the PDA culture medium, LJD-1 can grow at 23-38 ℃, and the optimal growth temperature is 28 ℃ of the enrichment temperature of the strain; the strain can grow under the pH condition of 4.0-9.0, and the optimal growth pH is 6.0; the bacteria have weak salt tolerance, can grow under the condition that the salt concentration is 0-3%, and grow best under the condition of no salt.

Example 3: experiment on degradation of phenanthrene

Preparation of microbial inoculum

1. Heating corn and water according to a mass ratio of 1:5 to prepare paste, and mixing the paste according to a mass ratio of 150: adding wood dust (200 mesh sieve), wheat bran and sodium lignosulfonate into the mixture at a ratio of 100:10:1, and kneading the mixture into a dough below the temperature. Placing the mixture of the bulk culture medium into a pill rolling machine to obtain spherical culture medium with diameter of 8mm, sterilizing, and oven drying.

2. And preparing the cultured LJD-1 fungus into a bacterial liquid with the hypha content of 10 g/L.

3. Adding the bacterial liquid into a sodium alginate solution with the mass fraction of 3% according to the mass ratio of 1:10, fully mixing the spherical culture medium and the solution, adding a sterile calcium chloride solution with the mass concentration of 4% after the mixture is finished, and hardening for 20min to obtain the pellet-shaped encapsulated fungi.

4. And (3) putting the encapsulated fungus pellets into a sterile culture bag, culturing in an incubator at 28 ℃ for 3-7 days until white hyphae grow on the surface, and thus obtaining the LJD-1 microbial inoculum.

Secondly, respectively inoculating the strain LJD-1 or LJD-1 microbial inoculum after activated culture for 7d into a strain containing 50 mg.L initially according to the inoculation amount of 10 percent^-1The phenanthrene was cultured in an inorganic salt medium (example 1, NaCl removed, pH 6.0) at 28 ℃ for 7 days with shaking, and 3 parallel experiments were performed. The treatment without adding pure bacteria Pyrromema domesticum LJD-1 and LJD-1 inocula is the control treatment.

Taking each processed sample for chemical analysis, and the specific steps are as follows: (1) sample pretreatment: adding dichloromethane into each culture sample for extraction, simultaneously adding 5 mu L of recovery rate indicator (phenanthrene-d 10) with the concentration of 200mg/L, fully shaking, and transferring into a separating funnel for standing. And collecting organic phases after layering, putting the lower layer liquid back to a shake flask, repeatedly extracting with dichloromethane of the same volume, combining the extracts, transferring the combined extracts to a flat-bottomed flask containing a proper amount of activated copper sheets for rotary evaporation, concentrating to about 2mL, adding a small amount of n-hexane (about 5mL), carrying out rotary evaporation to 2mL, repeatedly washing for three times, and replacing the organic solvent with the n-hexane. The concentrate after the displacement was purified by means of a glass-packed column (diameter: about 9 mm). The column packing was 3cm 3% deactivated neutral alumina, 3cm 3% deactivated silica gel and 1cm anhydrous sodium sulfate from bottom to top. Activating the column with an appropriate amount of n-hexane, rinsing the packed column with 15mL of mixed n-hexane/dichloromethane (volume ratio of 1:1), collecting about 15mL of eluate in a brown reagent bottle, blowing nitrogen to concentrate the eluate to about 0.5mL, transferring the eluate to a 1.5mL cell bottle, and freezing and storing the eluate. 5 mul internal standard hexamethylbenzene was added before the machine measurement, and the concentration was 200 mg/L. (2) Analyzing by an instrument: an Agilent 7890 gas chromatograph-5975 mass spectrometer is used for measuring the content of PAHs. The separation and analysis were carried out by means of an Agilent DB 5-MS (column length 30m, inner diameter 0.25mm, film thickness 0.25 μm) capillary chromatography column at a flow rate of 1.2 ml/min. The data obtained were processed using an agilent chromatography workstation and phenanthrene quantification was performed using a 6-point calibration curve and an internal standard method.

The strain LJD-1 and the microbial inoculum can degrade phenanthrene according to GC-MS determination and analysis, and the degradation rate can reach more than 60% after the strain is cultured in an inorganic salt culture solution containing phenanthrene with the concentration of 50mg/L for 7 days (figure 4). Wherein the degradation rate of the LJD-1 pure bacteria is 79.1 percent, and the degradation rate is 61.0 percent after the LJD-1 pure bacteria are prepared into a microbial inoculum. The strain LJD-1 is a powerful bacterium capable of degrading phenanthrene, and can also play a role in degrading phenanthrene after being prepared into a microbial inoculum.

And (4) conclusion:

1.1 phenanthrene degrading bacterium LJD-1 which can grow by taking phenanthrene as a carbon source is obtained from a soil sample obtained from a certain oil pollution site in Hangzhou through enrichment and separation.

2. The strain can form a colony with the diameter of about 8mm and growing upwards of white, round and white villous hyphae. The strain is an obligate aerobic strain. According to the analysis of a molecular biological means, the fungus LJD-1 separated in the experiment can be obtained as a Pyronema domesticum strain, and an evolutionary tree thereof is drawn. At present, few reports are made on the application of the strain, and particularly, no report is found on the research of degrading polycyclic aromatic hydrocarbon and phenanthrene by using the strain.

3. The optimal growth conditions for strain LJD-1 were a temperature of 28 ℃, pH 6.0, without addition of NaCl. LJD-1 can be degraded by using phenanthrene as carbon source at an initial concentration of 50 mg.L^-1After the inorganic salt culture solution is cultured for 7 days, the degradation rate can reach over 79 percent. After the strain LJD-1 is prepared into a microbial inoculum, phenanthrene can be efficiently degraded (the degradation rate is 61.0%). In conclusion, LJD-1 is a fungus capable of efficiently degrading polycyclic aromatic hydrocarbons, and the microbial inoculum prepared from the LJD-1 also has a good degradation effect and a good application potential in bioremediation of PAHs.

Sequence listing

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Claims (10)

1. Firecrackers (Zhaohuosi)Pyronema domesticum) LJD-1, accession number: GDMCC No: 61797.

2. use of the pyrophyllite LJD-1 according to claim 1 for degrading phenanthrene.

3. The use according to claim 2, wherein the degrading phenanthrene is degrading phenanthrene in petroleum contaminated soil.

4. The use according to claim 2, wherein the pyrothrix bricus LJD-1 is used in a phenanthrene-contaminated environment to degrade phenanthrene.

5. A phenanthrene-degrading bacterial agent, characterized by comprising the brewsonia fimbriata LJD-1 of claim 1 as an active ingredient.

6. The phenanthrene degradation microbial inoculum according to claim 5, wherein the preparation method of the phenanthrene degradation microbial inoculum comprises the following steps:

a. heating corn and water according to a mass ratio of 1:5 to prepare paste, and mixing the paste according to a mass ratio of 150: adding wood dust, wheat bran and sodium lignosulfonate at a ratio of 100:10:1, kneading into a dough at a low temperature, putting the mixture of the dough-shaped culture matrixes into a pill kneading machine to prepare spherical culture matrixes, and sterilizing and drying for later use;

b. preparing the brewsonia inermis LJD-1 into a bacterial liquid;

c. adding the bacterial liquid into a sodium alginate solution with the mass fraction of 3% according to the mass ratio of 1:10, fully mixing the spherical culture medium with the solution, adding a sterile calcium chloride solution with the mass concentration of 4% after the mixture is completely mixed, and hardening to obtain the pellet-shaped encapsulated fungi;

d. and (3) putting the encapsulated fungus pellets into a sterile culture bag, culturing in an incubator at 28 ℃ for 3-7 days until white hyphae grow on the surface, thus obtaining the phenanthrene degradation microbial inoculum.

7. The phenanthrene degradation bacterial agent according to claim 6, wherein the bacterial liquid in the step b is bacterial liquid with hypha content of 10 g/L.

8. A method for degrading phenanthrene, characterized in that the pyrothrix bricus LJD-1 of claim 1 is sprinkled in an environment containing phenanthrene to degrade phenanthrene.

9. The method according to claim 8, wherein the phenanthrene is degraded by spraying the pyrophyllum bricatum LJD-1 into a phenanthrene contaminated environment.

10. The method according to claim 9, wherein the phenanthrene is degraded by spraying the pyrophyllum bricatum LJD-1 into the petroleum contaminated soil.

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